Bestrophin 2 is expressed in human non-pigmented ciliary epithelium but not retinal pigment epithelium.

PURPOSE
Mice in which bestrophin 2 (Best2) is disrupted exhibit changes in aqueous flow and drainage, resulting in a reduction in intraocular pressure in comparison to wild-type mice. Best2 encodes a putative anion channel localized uniquely to the basolateral plasma membrane of non-pigmented epithelium cells in mice. In this study, we examine the localization of Best2 in the human eye.


METHODS
Rabbit polyclonal antibodies recognizing human Best2 (hBest2) were generated and characterized for use in western blot, immunoprecipitation, and immunofluorescence assays. The expression of hBest2 using these antibodies was examined using human donor eye tissues.


RESULTS
We could not detect hBest2 in human ciliary bodies or other ocular tissues by western blot. However, when enriched by immunoprecipitation, hBest2 was identified in ciliary bodies, but not in the retinal pigment epithelium. Using immunofluorescence, we located hBest2 in the basolateral plasma membrane of non-pigmented epithelial cells.


CONCLUSIONS
We found expression of hBest2 similar to mice only in NPE cells. These data suggest that Best2 may play a functional role in the regulation of aqueous flow and drainage in humans. We conclude that Best2 represents a new potential target for glaucoma therapy.

Bestrophin 2 is a member of the Bestrophin/RFP-TM family of proteins [1,2]. There are four paralogous groups of bestrophin genes in mammals, designated Best1 through Best4. In the mouse, Best4 is a pseudogene [3]. There is little information on BEST4 in any species [1]. Only Best1 and Best2 are known to be expressed in the eye [3][4][5]. In all species examined to date, Best1 is localized exclusively in the basolateral plasma membrane of the retinal pigment epithelium (RPE) cells [5][6][7][8][9]. The localization of Best2, however, is known only in the mouse [4]. Using mice in which the Best2 gene was disrupted by insertion of a Lac Z reporter, we found that Best2 gene expression is strongest in the nonpigmented epithelium (NPE) cells of the eye and in colon epithelia [4]. Antibodies specific to mouse Best2 (mBest2) confirmed these findings and showed that mBest2 is localized to the basolateral plasma membrane of those cells. mBest2 is also expressed in the olfactory epithelium [10,11] and in salivary acinar cells [1].
Mutations in BEST1 are causally associated with five human retinal degenerative diseases [1,17]. Although mutations in Best2 are not known to cause disease, the Best2 null mouse was found to have a significantly reduced intraocular pressure (IOP) when compared to heterozygous and wild-type littermates [4]. In a follow-up to that work, we demonstrated that lack of Best2 results in an increase in aqueous flow and in drainage via the conventional and uveoscleral pathways [18]. Recent work in our laboratory and others has linked bestrophins to regulation of voltagedependent Ca 2+ channels [14,[19][20][21], and has found a significant permeability of Bestrophin channels to bicarbonate [22]. The latter function could explain the phenotype of the Best2 null mouse, and explain the apparent synergistic effect on IOP of carbonic anhydrase inhibitors and lack of Best2 [4]. This phenotype suggests that Best2 could be an attractive target for diminishing IOP in individuals with glaucoma. However, little is known about Best2 in humans. As such, our goal in this study was to determine whether hBest2, like mBest2, is exclusively located in NPE cells in the eye.
inhibitor cocktail III (1:100; EMD Chemicals, Gibbstown, NJ). Lysates were precleared with 200 μl of Pansorbin A (Calbiochem) for 1 h at 4 °C and centrifuged at 10,000× g for 5 min. The supernatant was split into 2×5 ml samples. One was incubated with 20 μl of GA3512 coupled to sephrose 4B protein A beads (ZYMED, San Francisco, CA). The other sample, a control, was incubated with uncoupled sephrose 4B protein A beads. After 1 h at 4 °C the beads were recovered by centrifugation at 5,000× g, and washed 5× in lysis buffer and 1× in 50 mM Tris, pH 8.0. Beads were then resuspended in 50 μl of Laemmli sample buffer, incubated for 5 min at 95 °C, and resolved on 10% SDS-PAGE gels. Following transfer to polyvinylidene fluoride (PVDF) membranes, Bestrophins were identified by western blotting as described previously [5]. For controls, lysates of HEK293 cells or transfected HEK293 cells expressing hBest2 were immunoprecipitated simultaneously. Immunofluorescence: Human donor eyes were obtained from the Lions Eyebank of Oregon. Postmortem collection times were under 6 h. Anterior segments were dissected from each globe and fixed in 4% paraformaldehyde in phosphatebuffered saline. Following cryoprotection of the tissue in 30% sucrose in phosphate-buffered saline, the tissue was embedded in Tissue Tek Optimal cutting temperature (VWR, Batavia, IL) and 10 μm sections were cut using a cryostat. HEK293 cells grown on coverslips and transfected with pAdlox-hBest2 or cryosections of human anterior segments were stained using the GA3512 antibody as described previously [25] with goat anti-rabbit Alexa Fluor 568 (Invitrogen, Eugene, OR) as a secondary antibody. In some experiments, human anterior segments were also stained with monoclonal antibody 9A5, recognizing the α1 subunit of Na + /K + ATPase. Nuclei were labeled with 4,6'-diamidino-2phenylindole (DAPI; Sigma). Figure 1A, affinity-purified GA3512 recognizes recombinant hBest2 in lysates from transfected HEK293 cells, as shown by western blot. It also recognizes mBest2, though not with the same efficacy. To control for the possibility of cross-reaction with other bestrophins, we included recombinant hBest1, mBest1-EGFP, and mBest3 on our blots as well. Expression of these other bestrophins was confirmed by western blot (Figure 1D-F). GA3512, does not crossreact with human or mouse Best1 or Best3 ( Figure 1A). We compared the specificity of GA3512 with a commercial rabbit anti-hBest2 polyclonal antibody (NOVUS, Biologicals, Littletown, CO; Figure 1B). The Novus antibody also was Figure 2. Immunoprecipitation of hBest2 from human donor tissues. A: Lysates of human ciliary bodies (hCBs), human RPE (hRPE) cells, HEK-293 cells (HEK control), or HEK-293 cells transfected with padlox-hBest2 (HEK w/hBest2) were immunoprecipitated with GA3512 coupled to protein A beads or control beads and blotted back with GA3512. hBest2 was identified in hCB and HEK w/hBest2 when immunoprecipitated with GA3512 beads, but not control beads. B: Extraneous bands in A were also present in blots when primary antibody was omitted, and likely represent IgG that has leached from the GA3512-coupled beads. specific to Best2, but like B4947A ( Figure 1C), which was raised against mBest2, it was more efficient at identifying mBest2 than hBest2.

As shown in
Having demonstrated the specificity of GA3512, we next tried to identify hBest2 in human ocular tissues. However, we could not identify the protein by western blot using human tissue lysates (not shown). To determine whether this was because of low levels of expression, we immunoprecipitated hBest2 using GA3512 covalently coupled to Protein A. As shown in Figure 2A, GA3512 specifically detected hBest2 in ciliary bodies and in transfected HEK293 cells, but not in human RPE cell lysates or untransfected HEK293 cells. The extraneous bands present in the immunoprecipitates were recognized by the secondary antibody used in western blotting ( Figure 2B) and presumably represent IgG leaching from the GA3512-coupled beads. We conclude that hBest2 is expressed in human ciliary bodies but not in the human RPE.
We next used GA3512 to determine the localization of hBest2 in human ciliary bodies. As a precursor to these experiments, we performed immunolocalization of hBest2 in transfected HEK293 cells. GA3215 specifically detected hBest2 in the plasma membrane of transfected HEK293 cells ( Figure 3A), and did not label untransfected HEK293 cells ( Figure 3B). To localize hBest2 in human ciliary bodies, we stained cryosections of human anterior segments with GA3512. Using DIC optics to identify various tissues in the Figure 4. Co-localization of hBest2 with the α1 subunit of Na + /K + -ATPase at the basal plasma membrane of the RPE. A: Cryosections of human anterior segments were reacted with GA3512 to identify hBest2. B: The localization of Na + /K + -ATPase was determined using a monoclonal antibody against the α1 subunit. C: Nuclei were stained with DAPI. D: A merge of the images shows that hBest2 co-localizes with Na + /K + -ATPase at the basal surface of the NPE. note that the α1 subunit of Na + /K + -ATPase is also found in the plasma membrane of PE cells, where hBest2 is not expressed. eye ( Figure 3D), we determined that specific labeling for hBest2 was confined to NPE cells in human ciliary bodies ( Figure 3C). Although the orientation of the cells varied in several cells, there was a clear separation of hBest2 staining and the DAPI-stained nuclei, suggesting its localization to the basolateral plasma membrane of the NPE. No staining was observed in control sections in which the GA3512 antibody was omitted ( Figure 3E).
To more precisely localize Best2, we stained human anterior segments with GA3512 and monoclonal antibody 9A5, which recognizes the α1 subunit of Na + /K + ATPase, a marker for human ciliary epithelial plasma membranes. Inspection of the tissue using a 100× oil immersion objective (na=1.3) revealed that Best2 is predominantly located in the basal membrane of the NPE. In contrast, and as previously reported [26,27], Na + /K + ATPase stained the plasma membranes of both the pigment epithelium and NPE cells. Colocalization of Best2 and Na + /K + ATPase occurred at the basal membrane of the NPE, but nowhere else.

DISCUSSION
In this study, we sought to determine the location of hBest2 in the human eye. To accomplish this, we generated a novel rabbit anti-hBest2 IgG (GA3512) that is specific to Best2 ( Figure 1) and preferentially recognizes hBest2 over mBest2. GA3512 demonstrated a high specificity and titer in western blotting, immunolocalization, and immunoprecipitation assays.
Having validated the specificity of GA3512, we tried to determine whether hBest2 is expressed in the NPE, as mBest2 is in the mouse. We have previously shown by RT-PCR that hBest2 mRNA is found in human ciliary bodies [4]. Unfortunately, western blotting proved unsuccessful at identifying hBest2 in any ocular tissue. We hypothesized that this was due to a low level of hBest2 protein expression, and attempted to enrich hBest2 by immunoprecipitation. This proved successful, and we were able to identify hBest2 in immunoprecipitates from human ciliary bodies. We did not observe hBest2 in immunoprecipitates from human RPE cells ( Figure 2). We next used GA3512 to determine the specific location of hBest2. As shown previously for mBest2 [4], in transfected HEK293 cells, hBest2 is localized within the plasma membrane ( Figure 3A). Immunofluorescence staining of human donor eyes identified hBest2 staining only in the ciliary bodies ( Figure 3C), where it appeared to be uniquely expressed in NPE cells. We sought to more precisely locate hBest2 in the NPE by comparing its localization in the α1 subunit of Na + /K + -ATPase (Figure 4). Similar to previous reports [26,27], we found that in human ciliary bodies, the α1 subunit of Na + /K + -ATPase is localized in the basolateral plasma membrane of both PE and NPE cells ( Figure 4B,D). In contrast, hBest2 staining co-localized with Na + /K + -ATPase only at the basolateral plasma membrane of NPE cells ( Figure   3 and Figure 4A,D). This is the same location that we have previously shown for mBest2 in mice [4].
The phenotype of Best2 −/− mice is a diminished IOP [4,18]. Determination of aqueous dynamics in Best2 −/− mice indicates that aqueous flow, however, is increased. The diminished IOP results from overcompensation of the outflow pathways [18], indicating that in the mouse, Best2 plays a role in regulating IOP by affecting both inflow and outflow. The findings reported herein, that hBest2, like mBest2, is located in the basal plasma membrane of NPE cells in humans, suggests that Best2 may also be an important player in regulating IOP in humans. Consequently, these data support the hypothesis that modulation of Best2 activity may represent a new therapeutic avenue for lowering IOP in individuals with glaucoma.

ACKNOWLDEGEMENTS
Work in the author's laboratories is funded by grants from the NIH (EY13160), Alcon Research Laboratories, the American Health Assistance Foundation, and an unrestricted grant to the Department of Ophthalmology and Vision Science at the University of Arizona from Research to Prevent Blindness.